Notch1 activation and TGF-beta stimulation in transformed human esophageal cells

Transformed human esophageal keratinocyte cell line EPC2-T (EPC2-hTERT-EGFR-cyclin D1-p53R175H) cells were stimulated with or without 2.5 ng/ml recombinant human TGF-beta1 for 10 days. The above cells were subjected to treatment for 10 days with or without 0.5 µg/ml doxycycline (DOX) to activate tetracycline-inducible (tet-on) ICN1, an active form of Notch1. Transformed human esophageal keratinocytes EPC2-T (EPC2-hTERT-EGFR-cyclin D1-p53R175H) were stably transduced with tetracycline-inducible ICN1 (active form of Notch1). Cells were treated for 10 days either with or without 2.5 ng/ml recombinant human TGF-beta1 (TGF) along with or without 0.5 µg/ml doxycycline (DOX). Then, cells were subjected to RNA extraction in triplicate for gene array experiments. Four conditions were compared: (1) neither TGF-beta1 treatment nor Notch1 activation (cont); (2) Notch1 activation only (DOX); (3) TGF-beta1 stimulation only (TGF); (4) Notch1 activation plus TGF-beta1 stimulation (DOXTGF).

转化型人食管角质形成细胞系EPC2-T（EPC2-hTERT-EGFR-cyclin D1-p53R175H）分别在有、无2.5 ng/mL重组人转化生长因子β1（TGF-β1）的条件下刺激培养10天。上述细胞分别在有、无0.5 μg/mL多西环素（DOX）的条件下处理10天，以激活四环素诱导型（tet-on）ICN1——Notch1的活性形式。将转化型人食管角质形成细胞EPC2-T（EPC2-hTERT-EGFR-cyclin D1-p53R175H）稳定转染携带四环素诱导型ICN1（Notch1的活性形式）的载体。将上述细胞分别在有、无2.5 ng/mL重组人转化生长因子β1（TGF-β1，简称TGF）以及有、无0.5 μg/mL多西环素（DOX）的组合条件下处理10天。随后，对细胞进行三次生物学重复的RNA提取，用于基因芯片实验。本实验共设置并比较以下4组实验条件：(1) 既不施加TGF-β1处理也不激活Notch1（对照组，cont）；(2) 仅激活Notch1（DOX组）；(3) 仅施加TGF-β1刺激（TGF组）；(4) 同时激活Notch1并施加TGF-β1刺激（DOXTGF组）。